The goal of this application is to elucidate the mechanisms of adenosine receptor (AR) signaling in the control of coronary vascular tone (CVT). There is a critical need to fully understand the control of CVT by adenosine (Ad) in normal and diseased hearts. We have achieved the major goals of the last renewal and made tremendous progress on how Ad interacts with the coronary artery (CA) smooth muscle cells (SMC) and endothelium (E) to produce relaxation using the single AR KO's (except A2B). We have shown that CASMC and E contain both A2A and A2B relaxing ARs; and CASMC and E also contain A1 and A3 contracting ARs. We established a major role for A2A and a minor role for A2B in the relaxation of CA and the involvement of A1 and A3 in the contraction of CA. Also, we established that E plays a role in releasing mediators (NO and COX-I products) that play opposite roles in modulating the action of Ad through 4 ARs. A1 AR in CASMC involves activation of PLC-2III-> PKC1 -> p42/44 in causing contraction. A2B causes relaxation thorough p-38 MAPK. However, we have observed an up-regulation of A2B in A2A and vice versa in single KO's. Thus, in order to avoid the compensatory phenotypic response of other ARs, we have created the double KO (DKO's, A2A/A2B and A1/A3) for functional and signaling experiments for this project. Our central hypothesis is that the control of CVT by Ad is determined by a net effect of relaxing (A2A/B, including E factors) and contracting (A1/3, including E factors) ARs. Binding of Ad to ARs may initiate signaling (Fig. A) through G proteins resulting in activation of distinct pathways to control CVT. This novel hypothesis will be tested with the use of innovative physiological genomic approaches using single and DKOs (A1/A3 and A2A/A2B). Use of DKO allows precise examination of AR-mediated signaling without the compensatory responses than through traditional approaches. Therefore, using single and DKO's, we propose 3 aims with isolated hearts, CASMC (+E cells) and aorta/mesenteric artery :(a) to characterize the relaxing and contracting phenotypic effects of Ad agonists/antagonists; (b) to identify the signaling involved in the action of Ad in coronary endothelial cells (release of NO from A2A andA2B (and A2A/A2B) and the coupling of A1 and A3 (and A1/A3) to PLA2; and the signaling in CASMC in A2A, A2B and A2A/A2B (PKA-p38-MAPK-HSP20) and in A1, A3 and A1/ A3 (PLC2III, PKC1, AKT and p42/44 ERK); and finally, (c) to investigate the roles of A1 andA3 in producing and the roles of A2A and A2B in inhibiting oxidative stress, respectively in the action of Ad. These studies have never been proposed before and, thus, the characterization of the heterogeneity of ARs and their signaling in the control of CVT by Ad using DKO are innovative and will move the field forward in two ways; (a) will have a positive impact in the field of vascular biology, in general, (b) will have a positive impact in the field of AR signaling, in particular. These studies will lead to the development of novel therapeutic agents for vascular disorders. Finally, our past work was instrumental in the approval of an A2A AR selective agonist (Lexican(R)) for myocardial perfusion imaging.